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SNP Detection summary
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A summary of the SNP detection course per week (6 weeks).
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- Find gene information from NCBI en ENSEMBL (week 1)o Know what is NCBI
National Center for Biotechnology Information
o Know what databases are in NCBI
1. GenBank primary sequence database
2. Pubmed (biomedical literature)
3. Entrez integrated molecular and literature databases
o Learn how to retrieve DNA sequences from NCBI
o learn to use the sequence searching tool BLAST to compare two gene sequences
o know the types of BLAST
1. BLASTp (protein blast)
2. BLASTn (nucleotide blast)
o know the parameters in BLAST
1. Search set
a. Default: non-redundant protein sequences (nr)
2. Choose organism
a. Default: all organisms
o Know the genes AMELX and AMELY
o Know what to expect with your AMELX and AMELY PCR experiment
o know how to locate a primer (set) in a gene sequence
o know how to calculate the size of your expected PCR product
1. Forward primer blast en reverse primer blast
2. (Highest number – lowest number) + 1 = PCR product
, - Primer parameters (week 2)
1. Ampliconlengte: qPCR: 100 bp; PCR: 500 bp
2. Product positie: dichtbij 3’ end
3. Tm product: Ta – 5
4. Ta product:
- Top hit altijd juiste antwoord?
o Know how to evaluate a BLAST output
E-value (aantal BLAST alignments je verwacht bij kans)
Hoe lager, hoe beter
Bit-score (kwaliteit van alignment)
Raw score: Identities + mismatches – gaps – ‘’-‘’
Hoe hoger, hoe beter (beste = 2* lengte van de query)
Een goede blast -> bit-score = 2x de lengte van alignment
Identity / similarity percentage (correcte hits)
o Know the statistics of E-value calculation while considering DNA
o Understand alignment penalty
1. Gap penalty = methode om 2 of meer sequenties te alignen.
a. Gaps zijn indels
b. Indels zijn uitzonderlijk i.v.m. substituties middels evolutie -> dus een gap maakt de
alignment zeldzamer
c. Als penalty value = laag -> niet-gerelateerde sequenties vormen een match met hoge
similarity scores
d. Penalty value = hoog -> gaps te moeilijk en slechte alignments
e. Empirische studie wijst uit dat een set van penalty scores leidt tot beste resultaten
o Understand the amino acid substitution table
Voor normale alignments geldt: iedere correcte alignment = 1 punt. Hoogste score = juiste
alignment. Voor proteïnen alignment is dit anders: alfabet van 4 -> 20 a.a.
1. PAM substitution matrix: iedere score geeft de kans dat een aminozuur van een rij wordt
vervangen door een aminozuur van een kolom.
a. Nadeel: PAM extrapoleert
2. BLOSUM matrix: iedere score is de log van de vervangingskans in conserved regions
a. Score reflecteert de frequentie waarop a.a. 1 en a.a. 2 elkaar zullen vervangen
BLOSUM80 PAM1 Proteïnen gerelateerd
BLOSUM62 PAM120 midrange
BLOSUM45 PAM250 Niet-gerelateerd
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